A common model of the mechanism of presynaptic and other intracellular membrane fusions is that the assembly of the SNARE core complex drives the approach and merger of the membranes that are to be fused. The assembly of the core complex has been studied primarily in solution and numerous features of intracellular membrane fusion have been inferred from analogies with viral fusion proteins. Therefore, several questions must be addressed in order to understand how presynaptic membrane fusion machines work at the molecular structural level. Are the SNARE assembly reactions the same on membrane surfaces as in solution? How and to what extent does the assembly of the SNARE core complex reduce the distance between the two membranes that are to be fused? How are the stoichiometries of t-SNARE receptors regulated on membrane surfaces and what are the reactive species? How is the force transduced from the complex into the two fusing membranes and what structural transformations of protein and lipid need to occur at the membrane surfaces to get two lipid bilayers to fuse? What are the roles of accessory proteins and specialized membrane lipids in this process? The goal of this project is to answer these questions by determining the general dynamic architecture of the SNARE complex as it forms on the surface of and in between membranes. Novel microscopic and spectroscopic approaches will be developed to achieve this goal and to test several sub-hypotheses of the general SNARE hypothesis of presynaptic membrane fusion. Four specific aims will be pursued: In aims 1 and 2, the dynamic architectures of various intermediates of SNARE complex assembly will be assessed in plasma membrane sheet and reconstituted membrane preparations, respectively; the role of cholesterol and phosphatidylinositol-4,5-biphosphate on SNARE complex assembly will be examined in specific aim 3; and, the structures of SNARE domains that are potentially important in force transduction will be determined in various lipid backgrounds in specific aim 4.